The field of the invention is synthesis of double-stranded DNA.
Conversion of a single-stranded ("ss") DNA template into a double-stranded ("ds") DNA molecule requires deoxynucleotide triphosphates ("dNTPs"), an enzyme capable of reading the template strand and incorporating the appropriate dNTPs into the complementary second DNA strand, and a primer able to provide a free 3' hydroxyl from which to start the second strand synthesis. This requirement for a primer has rendered difficult any attempt to synthesize a full-length second strand complementary to a linear template strand for which the nucleotide sequence at the 5' end is unknown, a problem that has been tackled in a variety of ways by researchers seeking to establish cDNA libraries (collections of cloning vectors into which have been cloned DNA copies of all or a selected fraction of the mRNA present in a sample of cells). Such libraries require the preparation of DNA species, termed "cDNA," complementary to all the mRNA species present in the cells. As described in detail by Maniatis et al. ("Molecular Cloning, A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982), preparation of ds cDNA from mRNA is a multistep process, beginning with the isolation of poly(rA).sup.+ mRNA from cells actively manufacturing proteins. [The term "poly(rA)" herein denotes an RNA homopolymer consisting entirely of adenosine ribonucleotide monophosphate (rAMP) units; "poly(dA)" would refer to a DNA homopolymer having adenosine deoxyribonucleotide monophosphate (dAMP) units. Each homopolymer made up of one of the other ribnucleotides or deoxyribonucleotides is herein represented in an analogous manner: e.g., "poly(rC)" is an RNA homopolymer made up of rCMP units; "poly(dC)" is a DNA homopolymer made up of dCMP units; "poly(rG)" is an RNA homopolymer made up of rGMP units; etc.]
The poly(rA).sup.+ mRNA species so isolated are then used as templates for the synthesis of cDNA, as follows: first, a synthetic poly(dT)-containing oligonucleotide is hybridized to the 3' poly(rA) tails of the mRNA molecules, where it serves as a primer for synthesis of the first strand of cDNA (sometimes termed the "antisense" strand) by a reverse transcriptase enzyme, using the mRNA as template. Under appropriate conditions, this enzyme can synthesize a full-length cDNA strand [i.e., a DNA strand complementary to the entire mRNA template, with the possible exception of some of the poly(rA) sequence at the 3' end of the mRNA], yielding a full-length ss cDNA hybridized to its mRNA template. These mRNA.cDNA hybrids may be directly cloned into vectors and used to transform host cells, but the low efficiency of this technique renders it "unsuitable for constructing large numbers of cDNA clones." (Maniatis et al., p. 221.) More frequently, cloning is accomplished using ds cDNA produced from the ss cDNA by any of several methods, including the following (illustrated in FIGS. 1-4, respectively):
(1) The mRNA-cDNA hybrid is first treated with alkali to hydrolyze the mRNA, yielding ss cDNA. As ss cDNA will occasionally form, by self-hybridization of a few nucleotides at or near the 3' end of the molecule, transient "hairpin loops" (see FIG. 1) capable of priming synthesis of the second strand (the "sense" strand) of DNA from the 3' hydroxyl of the first strand, incubation of ss cDNA with DNA polymerase and all four dNTPs (i.e., dATP, dTTP, dCTP, and dGTP) for long periods (e.g., 20 hr) results in the conversion of many, if not all, of the ss cDNAs into ds cDNAs; the ss DNA loop joining the two complementary strands of the ds cDNA molecules may be removed with an endonuclease (such as S.sub.1 nuclease) which specifically digests single-stranded DNA, yielding blunt-ended ds cDNA copies of the original mRNAs, minus varying amounts of each mRNA's 5' terminal sequence.
(2) Gubler and Hoffman (Gene 25:263-269, 1983) describe a procedure, illustrated in FIG. 2, in which a fragment of the original mRNA primes synthesis of the second DNA strand. The mRNA half of the mRNA.cDNA hybrid is randomly nicked by treatment with the enzyme RNase H, producing a number of 3' hydroxyl ends within the mRNA half of the hybrid, each of which can prime 5'- 3' DNA synthesis along the cDNA template. Nick translation by DNA polymerase generates a series of various-length DNA partial copies of the mRNA strand complementary to portions of the first cDNA strand. As the polymerase molecule synthesizing the DNA strand primed at the nick nearest to the 5' end of the original mRNA moves along the template, it degrades downstream mRNA fragments and nascent DNA strands, resulting in a ds hybrid having one full-length ss cDNA strand (the first cDNA strand) and a second strand that is partially varying lengths of mRNA (at the 5' end) and partially newly-synthesized DNA. In order to clone this molecule, the leftover mRNA portion is removed by residual RNase and the resulting ss DNA tail on the first strand is clipped off, leaving a blunt-ended ds cDNA missing some of the 5' terminal sequence of the original mRNA.
(3) Unlike the two methods described above, the method for ds cDNA synthesis reported by Land et al. (Nuc. Acids Res. 9:2251-2266, 1981) produces a ds cDNA copy of the entire mRNA, plus additional sequence that was not originally in that mRNA. The Land et al. method, illustrated in FIG. 3, begins with a ss cDNA from which the mRNA has been removed by alkaline hydrolysis. To the 3' end of this ss cDNA is added a poly(dC) tail, the synthesis of which is catalyzed by the enzyme terminal deoxyribonucleotidyl transferase (TdT). This homopolymeric tail at the 3' end of the first cDNA strand then serves as a site for hybridization with a complementary homopolymeric oligodeoxynucleotide primer, oligo(dG). Second-strand DNA synthesis proceeds from the 3' hydroxyl of this primer, yielding a ds cDNA consisting of not only the full-length sequence of the original mRNA, but also a poly(dC.dG) extension at one end. Variations on this method which have been reported include combining a poly(dA) tail with an oligo(dT) primer, or a poly(dT) tail with an oligo(dA) primer. Regardless of which set of complementary deoxynucleotide homopolymers is used, the resulting homopolymeric extension, an artifact of the method used to generate the second cDNA strand, remains an integral part of the ds cDNA throughout the cloning procedure.
(4) A fourth method, reported by Okayama and Berg (Molec. Cellular Biol. 2:161-170, 1982) and illustrated in FIG. 4, also introduces a poly(dG.dC) tail onto one end of the ds cDNA. In this method, a ss poly(dT) tail is enzymatically added to the 3' end of one strand of a linearized ds DNA vector. Poly(rA).sup.+ mRNA is hybridized directly to this poly(dT) tail, positioning the 3' hydroxyl of the vector's poly(dT) tail to prime the synthesis of the first cDNA strand along the mRNA template. The 3' end of the newly-formed cDNA strand is then tailed with poly(dC), and a ds linker containing a ss poly(dG) tail is added to form a bridge between the two ends of the vector (see FIG. 4). Following treatment of this construct with DNA ligase and removal of the mRNA portion of the molecule, DNA polymerase is employed to generate the second cDNA strand, using the poly(dG) vector tail as primer and the first cDNA strand as template. Finally, DNA ligase closes the ds cDNA/vector circle, yielding a recombinant DNA vector containing full-length ds cDNA with a homopolymeric dG.dC extension at the 5' end of the "sense" strand. A variation on this method was described by Heidecker and Messing (Nucleic Acids Res. 11:4891-4904, 1983).